In the American College of Medical Genetics and Genomics (ACMG) classification system stop codon (truncation) variants are usually considered to be pathogenic/likely pathogenic [1
]. Additionally, stop codon variants of the BRCA2
gene are frequent among all pathogenic variants, leading to a significant increase in the risk of breast and ovarian cancer. However, damaging variants of the C-terminal of the BRCA2
gene have not been investigated or are not considered pathogenic due to Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) classification [2
]. Among their criteria, they suggest that “a variant predicted to disrupt expression only of protein sequence downstream of position 3325 would be considered unlikely to be clinically important. Further functional and clinical studies are underway to refine risk, if any, for predicted nonsense or frameshift variants downstream of position 3326”. The BRCA2
protein has multiple roles besides the well-known DNA double-stranded break repair by homologous recombination, such as maintaining genome stability, including DNA replication, telomere homeostasis and cell cycle progression [3
]. These functions have been investigated by different assays but not all functions are available for exploration due to either technical or study limitations. The C-terminal of the BRCA2
protein contains interaction sites of RAD51 and multiple phosphorylation sites affecting their function [4
]. While, among these, S3291 probably has the highest impact in the BRCA2
–RAD51 interaction, a protein sequence of amino acids 3265–3330 of the BRCA2
protein was also reported to bind RAD51 [4
]. Additionally, a serine at the position of 3397 located more terminally from c.9976A>T (K3326*) is also a phosphorylation site, the function of which has been poorly investigated (https://www.phosphosite.org
(accessed on 30 December 2020)). Furthermore, the interaction of the BRCA2
C-terminal with RAD51 may be less significant for homology-directed repair (HDR) than for the protection of stalled replication forks, a relatively newly discovered and HDR-independent function of BRCA2
]. Stalled replication fork degradation occurs due to MRE11 nuclease in the lack of BRCA2
-mediated fork protection, in which its C-terminus has an essential role [7
]. Although the defect of this function of BRCA2
did not lead to cell survival change, the frequency of chromosomal aberrations was found to be increased [7
]. It was suggested that BRCA2
protein defective in maintaining fork stability and still proficient in HDR would be insensitive to Poly (ADP-ribose) polymerase (PARP) inhibitors, which specifically exploit the defect of double-strand repair [7
Previous literature data regarding the clinical relevance of BRCA2
c.9976A>T C-terminal stop codon variants have remained controversial, suggesting either a potential pathogenic role [8
] or no clinical significance [11
]. The BRCA2
c.9976A>T variant results in a stop codon at amino acid position 3326. Initially, it was considered pathogenic due to its nonsense coding nature, however, it was reclassified as non-pathogenic based on case–control studies [11
]. Among previously published literature, breast cancer risk was elevated for BRCA2
c.9976A>T carriers when compared to a control population in three reports [9
]. Studies investigating only ovarian cancer patients, except of Stafford et al. (2017) [16
], showed similar odds ratios (ORs). Interestingly, among familial pancreatic, lung and upper aero-digestive tract (UADT) cancer patients, c.9976A>T carrier status was associated with increased risk for developing cancer [8
Using basic classification rules, the c.10095delins GAATTATATCT variant, due to its nature (a combination of a deletion and an insertion leading to frame shift and consequently a premature stop codon, Figure S1
), can be regarded as pathogenic. However, due to its localization (terminal from 3326 position), it is usually considered as a benign variant. Indeed, in ClinVar database, 11 of the 15 entries interpreted this variant as benign/likely benign and four submitters considered it as a variant with unknown significance (VUS). Additionally, of the 11 studies reporting c.10095delins GAATTATATCT, the vast majority considered it as a VUS [18
] or clinically not important [21
] in breast/ovarian cancer patients, and a VUS in familial pancreatic cancer [22
], while it was interpreted as benign in ovarian cancer patients [23
]. Interestingly, in a study prioritizing variants in hereditary breast and ovarian cancer genes in patients lacking known BRCA
mutations, the c.10095delins GAATTATATCT variant was categorized as likely pathogenic based on co-segregation analysis (likelihood ratio 3.71) [24
Therefore, the aim of our study was to investigate the prevalence of BRCA2 C-terminal stop codon variants among our breast/ovarian cancer patients sent to germline BRCA1/2 gene testing and their co-segregation with clinicopathological parameters and study the loss of heterozygosity and allelic imbalance. An extensive literature review of an additional 122,209 cancer patients was also performed to assess the effects of the c.9976A>T variant on cancer risk.
The clinical relevance of BRCA2 C-terminal stop codon variants remains controversial. The BRCA2 c.10095delinsGAATTATATCT variant located at the 3′ end of the gene is considered to be non-pathogenic based on the ENIGMA classification system. There are literature data regarding its allelic frequency and clinical relevance. Despite its low prevalence in control populations and its relatively higher frequency in breast/ovarian cancer patients, based on our and others’ findings (LOH, allele imbalance, segregation and linkage data), this variant can be considered as clinically non-significant.
c.9976A>T, despite being a truncating variant, is usually classified as non-pathogenic based on case–control studies [11
]. Indeed, in our study, the disease onset, tumor proliferation index or other pathological and clinical parameters did not differ in carriers compared to pathogenic BRCA2
carriers or to BRCA1/2
wild type patients. Additionally, we did not find an increased prevalence among carriers or in carrier families for HBOC. The MAF of c.9976A>T is around 1% among patients that also counts against its independent pathogenic role. The lack of genotype–phenotype segregation, lack of LOH and lack of allelic imbalance in patients are all in line with previous literature [11
]. However, environmental factor-associated cancers (lung and skin carcinoma) were more frequent in families of the BRCA2
c.9976A>T carrier probands.
In previously published data, breast cancer risk was mildly elevated in BRCA2
c.9976A>T carriers when compared to control populations in three reports [9
]. Studies investigating only ovarian cancer patients, except that of Stafford et al. (2017) [16
], showed similar ORs. In the study of Stafford et al., in all cases, the germline c.9976A>T variant coexisted with other deleterious variants in other genes belonging to the BRCA2
pathway. Among familial pancreatic, lung and upper aero-digestive tract (UADT) cancer patients, the c.9976A>T carrier status meant high odds (4.24, 3 and 2.53, respectively) for developing cancer [8
]. In line with this, we observed an increased proportion of pancreatic cancer prevalence in families of double heterozygotes (c.9976A>T with pathogenic BRCA1
variant), however, due to the limited number of cases, this observation should be validated in a larger sample cohort. Additionally, regarding pancreatic cancer, further analysis is subject to bias due to the secondary assessment of datasets. Others also suggested that the concomitant c.9976A>T variant should be considered during genetic counseling for a potentially earlier age of HBOC cancer onset [16
]. In the study of Akbari et al. (2008), c.9976A>T carrier status was associated with a high OR of developing esophageal cancer (6.0; 95%CI: 1.3–28; p
= 0.01) [27
]. Higgs et al. (2015) also reported multiple co-occurrences of the BRCA2
c.9976A>T variant with the pathogenic BRCA2
c.6275_6276delTT (p.(Leu2092ProfsTer7)) frameshift variant in breast and ovarian cancer patients [13
]. The authors concluded that associations of increased cancer risk due to BRCA2
c.9976A>T represented a reporting bias and this was due to the variant being in linkage with BRCA2
c.6275_6276delTT. However, in our patient cohort, neither investigated C-terminal stop codon variant was associated with any pathogenic BRCA2
variant. Hence, we suggest that the linkage of the two BRCA2
variants can be a founder phenomenon in the investigated cohort reported by Higgs et al. [13
]. This is supported by other studies too [15
], therefore, the reported variant associations may be a population specific-phenomenon representing a founder effect.
Although, based on our findings and previously published data, the BRCA2
c.9976A>T variant alone probably cannot be considered as a risk factor for breast and ovarian cancer, it seems to be associated with other cancer types. Genetic epidemiological evidence suggested that the BRCA2
c.9976A>T variant contributes to the risk of developing familial pancreatic cancer [8
] and lung cancer [10
]. Additionally, it was reported that the risk of developing lung cancer is approximately doubled for smokers compared to non-smokers when carrying the c.9976A>T variant [17
]. Therefore, Wang et al. suggested that this finding may have implications for identifying high-risk ever-smoking subjects for lung cancer screening. Furthermore, it was reported [51
] that the c.9976A>T variant was associated with cancers that have strong environmental genotoxic risk factors. Based on functional studies, the authors proposed that the variant protein could probably retain the DNA repair capabilities important to hormone-responsive tissues but it might be less efficient in counteracting genotoxic stress [51
]. In line with this, based on associations between this BRCA2
variant and upper aero-digestive tract and lung cancer risk, PARP1 inhibitors were suggested as potential treatment strategies [17
]. These findings have not been confirmed by functional studies investigating the role of the c.9976A>T variant. Its damaging effects on the protein subcellular localization, cell viability, homology-directed repair (HDR) of double-strand breaks, centrosome amplification or sensitivity to DNA damaging agents [56
] were not observed. Moreover, it has been suggested that the protein, translated from the variant-carrier transcript, is defective in maintaining fork stability while being still proficient in HDR. Therefore, c.9976A>T carriers may be insensitive to PARP inhibitors, which specifically exploits the defect of double-strand repair [7
]. As a consequence, PARP-targeting therapy may not only be ineffective in these cases, but also induce further mutagenesis and genomic instability [7
]. All these findings indicate that the clinical value of the use of PARP inhibitors in BRCA2
c.9976A>T carriers should be further investigated.
In summary, the clinical phenotypes associated with C-terminal BRCA2
variants are significantly different from those observed in families with highly penetrant BRCA2
]. For the expected pathogenic BRCA2
mutation-associated cancer types (including breast, ovarian and prostate cancer), the C-terminal BRCA2
variants have not been found as risk factors [49
]. However, these variants may be involved in the pathogenesis of pancreatic and environmental factor-associated cancers.
4. Materials and Methods
4.1. Cases: Patients and Relatives
We investigated 2491 independent patients (probands) with breast and/or ovarian cancer sent for germline BRCA1/2
genetic analysis to the Department of Molecular Genetics at the National Institute of Oncology, Hungary between 2014–2019. Only one variant carrier per family, the proband, was included in our analysis. Among them, the BRCA2
C-terminal stop codon variants (LRG_293t1:c.9976A>T and/or c.10095delinsGAATTATATCT) were identified in 49 cases (average age: 43.4±10.1 years; 47 females, 2 males). Estrogen, progesterone, HER2 receptor status, Ki67 proliferation indices and histology were assessed as part of the routine diagnostics. All data were collected from the institutional medical information system. Details (patient characteristics and histology findings) are summarized in Table 1
. The study was approved by the Scientific and Research Committee of the Medical Research Council of the Ministry of Health, Hungary (ETT-TUKEB 53720-4/2019/EÜIG). Fisher’s exact test was used to examine the significance of the association (contingency) between phenotype and variant carrier status.
As a part of the Consortium of Investigators of Modifiers of BRCA1/2
(CIMBA) and the Breast Cancer Association Consortium (BCAC), standard phenotypic and epidemiological data collection was applied from 1998 during the study (http://cimba.ccge.medschl.cam.ac.uk/
(accessed on 30 December 2020); http://bcac.ccge.medschl.cam.ac.uk/bcacdata/
(accessed on 30 December 2020)). Details of data collection protocols have been used and reported previously [60
]. Accordingly, for the analysis of phenotypic and pedigree data, standard questionnaires (one is patient/disease-centered and one for pedigree data) were sent out to all patients in advance. Based on the standard data, acquisition pedigrees were generated. During genetic counseling, data reliability was confirmed by reviewing all medical reports available by practicing clinical geneticists. In the family history analysis, three-generation pedigrees were investigated where only the presence of tumor types were considered (not the number of cases in each family).
4.2. Nucleic Acid Extraction
Germline variants were analyzed using total DNA extracted from peripheral blood using a Gentra Puregene Blood Kit (Cat No.: 158389, Qiagen, Hilden, Germany) following the manufacturer’s instructions.
A GeneRead DNA FFPE Kit (Cat No.: 180134, Qiagen, Hilden, Germany) was applied to isolate genomic DNA from formalin-fixed paraffin-embedded (FFPE) tissues in an automated way using the QIAcube Instrument (Qiagen, Hilden, Germany).
RNA extraction was performed from total blood taken into Tempus™ Blood RNA Tubes (Thermo Fisher Scientific, Waltham, MA, USA) by a Tempus™ Spin RNA Isolation Kit. RNA quality and quantity were determined by a NanoDrop® 1000 Spectrophotometer (NanoDrop Technologies, Thermo Fisher Scientific, Waltham, MA, USA).
4.3. Genetic Analysis (Sequence and Copy Number Analysis by Next Generation Sequencing (NGS) and Multiplex Ligation-Dependent Probe Amplification)
Genetic analyses were done as we previously reported [62
]. Germline BRCA1/2
variant status was evaluated following library preparation using CE-IVD BRCA MASTR Plus Dx kit (Agilent, Santa Clara, CA, United States). Sequencing of the library was run on an Illumina MiSeq Instrument using MiSeq Reagent Kit v2 (500-cycles) (MS-102-2003, Illumina). Data analysis was done by MASTR Reporter software, a comprehensive CE-IVD marked (complies with the European In-Vitro Diagnostic Devices Directive) molecular solution for the identification of coding region variants in the BRCA1
genes. Copy number analysis was performed by the Multiplex Ligation Dependent Probe Amplification (MLPA) method using P002 and P239 probe sets for BRCA1
, and the P045 probe set for BRCA2
(MRC-Holland, the Netherlands).
Clinical significance of variants was evaluated and interpreted following AMCG/AMG recommendations [1
], ENIGMA classification [2
] and literature data mining.
The following transcripts were used for variant annotation. BRCA1: LRG_292t1 (NM_007294.3) and BRCA2: LRG_293t1 (NM_000059.3).
4.4. Sanger Validation and LOH Analysis
All germline pathogenic, likely pathogenic and variants of unknown significance (VUSs) were validated by traditional bidirectional Sanger sequencing on an independent blood sample. For loss-of-heterogeneity (LOH) testing, DNA from tumor tissues was used for PCR amplification by a Qiagen Multiplex PCR Kit (Qiagen). PCR product was purified by ExoSAP-IT™ reagents (Thermo Fisher Scientific, Waltham, MA, United States), then purified amplicons were sequenced bidirectionally on an ABI3130 Genetic Analyzer (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) using a BigDye™ Terminator v.1.1 kit (Thermo Fisher Scientific, Waltham, MA, USA).
4.5. Transcript Allelic Imbalance
Relative expression of the variant carrier and the normal allele was tested with Sanger sequencing. The ratio of electropherograms of the variant position on the cDNA template relative to the gDNA template was calculated. Briefly, cDNA was generated from 500 ng RNA using SuperScript™ IV Reverse Transcriptase (Thermo Fisher Scientific). cDNA primers were designed using Primer3Plus software (https//:primer3plus.com
): B2-C-e24_For—GATCCAGACTTTCAGCCATCTT and Rd_B2_Ex27.01_Rev—CGTCGTTTCAGTCTGAGATAATCT. Following PCR amplification and Sanger sequencing, data were visualized in Sequence Scanner software (Applied Biosystems, Thermo Fisher Scientific), and the peak ratio of the heterozygote position was given and compared to the peak ratio of the gDNA sequence of the same position for the same sample. The relative ratio was calculated and allelic imbalance was declared if the difference was >50%.
4.6. Statistical Analysis
For both proband characterization and family description, proportions and 95% confidence intervals by a modified Wald method were calculated using GraphPad QuickCalcs (https://www.graphpad.com/quickcalcs/confInterval1/
(accessed on 30 December 2020)). For statistical analysis, 2 × 2 contingency tables were applied and p
values were calculated by Fisher’s exact test. p
values were considered statistically significant at <0.05. Statistical power was calculated using the ClinCalc online algorithm (https://clincalc.com/stats/samplesize.aspx
(accessed on 30 December 2020)).
As a conclusion, our results suggest that among BRCA2 C-terminal stop codon variants, c.10095delinsGAATTATATCT is clinically non-significant. However, the c.9976A>T variant may have different clinical significance compared to the BRCA2 truncating variant before amino acid 3326. It may be considered as a genetic modifying factor in pancreas cancer when it co-occurs with pathogenic BRCA1 variants, although this observation should be validated in a larger sample cohort of double heterozygotes. Additionally, it seems to have an impact on the development of tumor types where environmental factors are significant as a genotoxic stress factor. Therefore, it is suggested to be a non-negligible variant, especially in the risk assessment of environmental cancers. The ACMG “pathogenic” classification is disease-specific. That is, a variant classified as (likely) benign with respect to HBOC still cannot be disregarded in conjunction with other, only loosely associated, diseases or with possible treatment options.
Additionally, our data, in line with a very recent review [63
], suggest that collecting disease-specific clinical data regarding C-terminal BRCA2
variants can assist in reducing the number of VUSs, which in turn may help in more precise treatment planning.